Our approach is to use molecular genetics to selectively and exclusively target the CA3 region allowing for a level of precision in our investigation that is not possible using more traditional pharmacological techniques. To this end, we have developed two transgenic lines of mice in which genetic manipulations are targeted to CA3 pyramidal neurons. In the first (CA3-NR1-KO), we have eliminated the essential NR1 subunit of the NMDA receptor, one of major brain excitatory receptors. Functional elimination of this receptor should alter the ability of CA3 neurons to undergo plastic changes in response to neural input and, in turn, alter the properties of the recurrent CA3 network. In addition, this receptor is believed to be critical for mediating the structural changes observed in response to chronic stress. We have two projects to investigate the role of CA3 in the pathology of epilepsy and chronic stress using these mice (Projects 1 and 2). In the second line of mice (CA3-DTR), we have introduced a diphtheria toxin receptor that is exclusively expressed in CA3. This allows us to systemically inject diphtheria toxin (DT) which has no effect on control mice but will selectively and inducibly eliminate pyramidal neurons in CA3. Using this genetically controlled method to eliminate CA3 at various time points following learning, we are able to address the specific role of this region in the development of long-term hippocampal-dependent memory (Project 3). Each of these projects is designed to answer questions that require a precisely controlled manipulation of a specific subregion in the hippocampus. As such, we expect to be able to provide critical new information about the role of CA3 in maintaining hippocampal integrity under pathological conditions as well as how this region contributes to memory formation in normal conditions. Project #1: Lack of Kainic Acid-Induced Gamma Oscillations Predicts Subsequent CA1 Excitotoxic Cell Death. We have investigated functional changes in the hippocampus in the CA3 network using CA3-NR1 KO mice (mutants). Electrophysiological recordings from CA1 revealed abnormally high amplitudes of oscillatory activity in the local field potentials during periods of awake immobility in the mutants. These results led us to predict that the mutants are more susceptible to epileptic insults. Using a well-established model of epilepsy, we found that an intraperitoneal injection of the excitotoxin kainic acid (KA) induced more severe behavioral seizures in the mutant mice than in their control littermates. Moreover, degeneration of CA1 pyramidal cells and GABAergic interneurons was observed in KA-injected mutants, while rarely observed in control genotypes. These results suggest that the mutants, in which CA3 neurons are lacking an NMDA receptor, are more susceptible to KA-induced seizures and neurodegeneration. To investigate how CA3 may be contributing to this exaggerated response, we recorded neural activity in CA1 after KA treatment and observed persistent 30-50 Hz gamma oscillations in control mice prior to the first seizure discharge that were absent in the mutants. Consequently, on subsequent days, mutants manifested prolonged epileptiform activity and massive cell death of both pyramidal cells and local GABAergic interneurons in CA1. However, pretreatment with -dendrotoxin to enhance presynaptic release of the inhibitory neurotransmitter GABA, maintained the gamma oscillations, diminished epileptiform activity, and prevented CA1 cell death in the mutants. This result indicates a crucial role for CA3 in modulating the inhibitory network within the hippocampus and controlling levels of excitability in pathological conditions. Clinically, these persistent gamma oscillations are frequently observed in human epileptic patients, especially before and at the onset of seizure discharges. Our results provide evidence that emergent low frequency gamma oscillations are negatively correlated with KA-induced CA1 cell death, thereby shedding light on the functional role of gamma oscillations in clinical epilepsy. Our results also suggest that the CA3 network is one of the critical areas in the generation of limbic seizures. Project #2: Effects of chronic stress on the hippocampus. Chronic stress leads to the development of a number of neurostructural, neuroendocrine and behavioral changes that may also precipitate the onset of many neuropsychiatric disorders. In animal models, one of the most dramatic effects of chronic stress in the brain is dendritic atrophy in the CA3 region of the hippocampus. It is not yet clear whether this structural change to the input region of CA3 neurons plays a causal role in the development of cognitive deficits associated with stress-related disorders or is an adaptive response that serves to minimize further damage to the hippocampal circuit. First, we confirmed that stress-induced atrophy does require a functional NMDA receptor as the CA3-NR1-KO mice were immune to the structural changes observed in control mice following chronic immobilization stress (CIS). Further, this CA3-specific genetic manipulation also prevents stress-induced atrophy in CA1, demonstrating a feed-forward effect of CA3 atrophy on the CA1 subregion of the hippocampus. These data indicate that the stress-induced changes in CA3 neurons may be pivotal in regulating the response of the hippocampus as a whole under conditions of chronic stress. The stress-induced increase in anxiety appears to be independent of morphological changes in hippocampal neurons as it was observed in both mutant and control mice. However, mutant mice exhibited impaired memory in a test of hippocampal-dependent short-term spatial memory. This result suggests that dendritic atrophy may be a compensatory mechanism that effectively maintains functional capacity within the intra-hippocampal network. Project #3: Role of CA3 in mediating consolidation of hippocampal-dependent memory. It has been proposed that CA3 is responsible for generating specific patterns of neural activity in CA1 that may play a role in coordinating hippocampal-cortical interactions critical for the long-term maintenance of certain forms of memory. Consolidation of memory, or the gradual stabilization of initially labile memory into a long-term memory trace, occurs over a period of time following initial learning. We hypothesized that the CA3 region of the hippocampus may be important in this time-dependent process whereby some types of short-term memories are transformed into stable long-term memories. Using the CA3-DTR mice, we were able to induce a neurotoxic lesion of CA3 following acquisition of a hippocampal-dependent task to investigate the role of this region in memory consolidation. Ablation of CA3, 24 h after training - during the proposed period of system-level memory consolidation, results in a loss of context-specific information about remotely acquired memories. Specifically, our data show that mice in which the CA3 region was lesioned shortly after experiencing a fearful situation were unable to distinguish specific details of the episode when tested approximately one month later. Thus, these data suggest that CA3 is critically involved in the development of long-lasting, accurate memories and may provide a basis for future investigations into the role of this brain region in memory-based disorders.